The present invention relates generally to immunogenic peptide-carrier protein conjugates, and particularly to conjugates comprising selected fragments of bacterial proteins and larger carrier proteins, which are useful in providing immunity to bovine respiratory disease.
Pneumonic pasteurellosis, also known as shipping fever, is a widespread cause of mortality and morbidity of feedlot cattle in North America. Affected cattle develop a fibrinous pneumonia marked by fever, severe pulmonary inflammation, accumulation of a fibrin-laden exudate in the lungs and pleura, and necrosis of alveolar tissue. Pneumonic pasteurellosis is the central component of the multifactorial syndrome known as bovine respiratory disease (BRD), in which stress and viral infection interact to compromise normal host defenses and enable bacterial colonization of the deepest regions of the bovine lung. In North America, the annual economic losses attributable to BRD via direct deaths, weight losses, and reduced productivity have been estimated to range between $200 and $500 million.
Pasteurella haemolytica (P. haemolytica), the etiologic agent of pasteurellosis, is a normal bacterial component of the upper respiratory tract of cattle. Under normal conditions, inhaled organisms reaching the lung encounter local cellular immunity provided by resident or elicited alveolar macrophages and neutrophils. During the course of bovine respiratory disease, however, a viral infection, combined with stress attributable to handling of feedlot cattle, results in immunosuppression, allowing P. haemolytica bacteria to gain access and multiply in the deep regions of the lung.
Attempts to control the disease by prophylactic or therapeutic means have been largely unsuccessful. Vaccination with killed P. haemolytica bacterins provides no protection against pneumonic pasteurellosis in cattle, nor does such vaccination reduce lung lesions in calves challenged experimentally by intraalveolar aspiration of live P. haemolytica. In contrast, vaccination with live P. haemolytica or culture supernatants has been shown to reduce the severity of pasteurellosis lesions in challenged calves. Such results suggest that live bacteria secrete a factor capable of inducing partial immunity to the effects of pasteurellosis.
Actively growing cultures of P. haemolytica elaborate an exotoxin capable of killing bovine leukocytes. This cytotoxin, or leukotoxin, is optimally produced by log phase cultures and is not evident in stationary phase cultures of the bacteria. Since leukotoxin is only elaborated by living bacteria, killed bacterins prepared from washed bacteria would not contain the toxin and would not, therefore, induce protective immunity against the toxin.
Partial purification of P. haemolytica leukotoxin has been reported by Himmel et al., Am. J. Vet. Res. 43:764 (1982), and Chang et al., Am. J. Vet. Res. 47:716 (1986). Ultrafiltration, gel filtration and polyacrylamide gel electrophoresis have indicated leukotoxin to be a 100-150 MW protein. Leukotoxin activity is O.sub.2 stable, but exquisitely heat sensitive, which has as yet precluded total purification of leukotoxin. Lacking homogeneous protein, sequencing of leukotoxin is impossible, rendering molecular manipulation at the gene level difficult.
Purification of P. haemolytica leukotoxin to a single protein moiety and elucidation of its amino-terminal sequence has enabled construction of immunogenic conjugates comprising leukotoxin N-terminal peptide fragments covalently joined to suitable carrier proteins. When administered to a subject animal, these conjugates are capable of eliciting antibody which specifically neutralizes the toxic effect of the P. haemolytica exotoxin on bovine leukocytes.